1. Field of the Invention
The present invention relates to methods and reagents for enumerating cells in samples of whole blood, and more particularly to methods and reagents for quantitatively determining reticulocyte and/or platelet levels in a whole blood specimen by fluorescence using a flow cytometer.
2. Description of the Prior Art
Blood is a fluid, circulating tissue found in all higher animals and in many invertebrates. It is a tissue, just as skin, muscle, and bone are tissue, because it contains living cells and has specific functions, chief among which being the conveyance of materials from one part of the body to another. The general principal on which the chemical life of an organism is conducted is that each living cell carries out within itself all the chemical processes necessary to its existence. Therefore, all of the materials required by each cell must be carried to it and all of the wastes must be removed. Throughout the bodies of higher animals, a highly specialized system of transport, called the blood vascular system, has evolved which affords an efficient route for the blood and provides the necessary intimate contact thereof with every living cell.
The principal materials which a living cell requires are sugar, amino acids, fats, vitamins, oxygen, salts, hormones, and water. The organs of digestion convert the solid constituents of food into forms that the blood can absorb and deliver to the cells of the body. The principal wastes which must be removed are carbonic acid and simple soluble compounds of nitrogen.
In all the higher animals, blood consists of an aqueous fluid part (the plasma) in which are suspended corpuscles of various kinds: the red blood cells (erythrocytes), the white blood cells (leukocytes), and the blood platelets. Plasma has a composition comprising roughly 90% water, 9% protein, 0.9% salts, and traces of other materials such as sugar, urea, uric acid, and the like. Plasma, in fact, resembles nothing so much as the primordial ocean in which the first unicellular animals developed.
The concentration of salts in plasma is important principally to assist dissolution of proteins. Most proteins will not dissolve in pure water and are therefore essentially unusable by living material without salts.
The normal concentration of hydrogen ions in plasma is 0.4.times.10.sup.-7 grams of hydrogen per liter, for a pH of 7.4. Because of the adverse affects of substantial variation from this level, the body acts to maintain the blood pH at 7.4.
The cells or corpuscles of the peripheral blood (i.e., the blood outside the bone marrow) are divided into two main groups: the red blood cells (erythrocytes), whose primary object is to transport oxygen, and the white blood cells (leukocytes), whose primary functions relate to the immune system and the destruction of materials foreign to the body. In addition to these two main groups, the blood also contains the so-called blood platelets, which are important in hemostasis.
The final stages of erythrocyte maturation occur after their release from the bone marrow while these cells are circulating in the peripheral blood. These young red cells, or "reticulocytes" have lost their nucleus and thus their ability to divide or to synthesize RNA. Although these functions have ceased, reticulocytes are still metabolically active and are capable of synthesizing protein, taking up iron for the synthesis of heme, and carrying out the necessary metabolic reactions required to maintain an energy rich state. These cells are usually distinguished from mature erythrocytes through the presence of the reticulum, which give them their name. This reticulum may be dyed by such agents as brilliant cresyl blue, nile blue sulfate, or new methylene blue, after which quantitation of reticulocytes may be performed by way of manual observation under a microscope.
Although reticulocytes normally comprise about one percent of the total red blood cell population, this percentage can change dramatically under abnormal conditions. For example, reticulocyte counts have been used for many years as a diagnostic aid in studying blood dyscrasias and as an index of red cell regeneration following hemorrhage, as well as for monitoring early toxicity in chemotherapy of certain malignant diseases.
Blood platelets are also produced in the bone marrow and are important in hemostasis, both in the formation of the primary hemostatic plug and in the intrinsic coagulation mechanism. Platelets are usually from one to four microns in diameter and circulate in the blood as smooth, disc-shaped cells having a circulating life span of approximately nine to eleven days. The measurement of platelet concentration is an important diagnostic aid in studying disorders of the blood coagulation system.
The enumeration of platelets is usually performed at the present time either by manual phase contrast microscopy or by automated systems based on electroconductometric or optical light scatter measurements. However, each of these techniques has its own disadvantages. With the manual phase contrast microscope, counting becomes extremely tedious, which could of course have significant effects on both accuracy and precision. Any manual counting technique places severe limits on the size of the population of cells which can be counted. Moreover, the smaller platelets are often masked by the larger red blood cells. An underlying problem with either of the automated methods is the inability to obtain conditions having a high signal to noise ratio. Thus, it is difficult to obtain sufficiently accurate measurements apart from background. Moreover, these measurements have an inherent inability to distinguish giant platelets from small erythrocytes. Other problems are presented by the currently-used automatic apparatus, such as clogging with the electroconductometric apparatus and the requirement of especially pure reagents to minimize microscopic debris for optical scatter apparatus.
The use of fluorescing stains or dyes for analysis of blood cells has been known for many years. For example, J. B. Vander, et al., J. Lab. Clin. Med 62, 132 (1963) described the use of the dye acridine orange for the identification of reticulocytes by fluorescent microscopy. However, this technique still requires visual examination of the sample and thus possesses the inherent disadvantages of such manual optical examination methods.
Additionally, many different types of automatic apparatus have been disclosed for detecting and quantitating blood cells, particularly white blood cells. Representative of such methods (some of which use acridine orange or other fluorescent dyes) are U.S. Pat. Nos. 3,497,690; 3,916,205; 3,864,571; and 4,027,971. While these references generally disclose the use of fluorescent dyes in a variety of apparatus, including a flow cytometer, they still fail to solve the problems of the prior art and do not provide a method or composition for quantitating reticulocytes or platelets by fluorescence.
Of particular interest in this regard are U.S. Pat. Nos. 3,684,377 (Adams and Kamentsky) and 3,883,247 (Adams). These patents relate to methods and dye compositions for quantitating cells (particularly white blood cells) using a metachromatic fluorochrome dye such as acridine orange.
The Adams and Kamentsky '377 patent describes the use of a vital dye composition for differential blood analysis of living white cells which consist essentially of acridine orange having a concentration between 10.sup.-7 and 10.sup.-5 grams per liter, the acridine orange solution having a pH factor and an osmolality within the normal physiological ranges for human blood plasma. While this '377 patent teaches that this composition is useful for identifying the various types of white blood cells and for distinguishing them from other bodies in the blood, there is no teaching that this composition has any use in the enumeration of reticulocytes or platelets.
The Adams '247 patent represents a modification of the teaching of the Adams and Kamentsky '377 patent in that the white blood cells are treated under conditions in which the cells are "shocked" by exposure to a non-physiologic medium during staining. That is, the staining composition used in the Adams '247 patent is made hypotonic, the osmolality or salinity thereof being generally below that normally found in human blood. The teaching of the Adams '247 patent is that this hypotonic condition produces a differential rate of uptake of acridine orange dye by the various types of white blood cells, thus allowing them to be more clearly distinguished from one another than in previous techniques. However, as with the Adams and Kamentsky '377 patent, the Adams '247 patent does not teach the quantitation of platelets. Moreover, although the Adams '247 patent does purport to disclose a method for the detection of reticulocytes, the method disclosed has been found by the present applicant to be practically useless for the quantitation of either reticulocytes or platelets, as further described below.
Accordingly, there exists a need for methods and reagents useful for quantitating reticulocytes and platelets by fluorescense using a flow cytometer. It is a principal object of the present invention to provide such reagent and methods.